Evaluation of the Antifungal Potential of Grape Cane and Flesh-Coloured Potato Extracts against Rhizoctonia sp. in Solanum tuberosum Crops

Potato (Solanum tuberosum) is one of the most important food crops worldwide, and Rhizoctonia solani infection is one of the most common diseases. The objective of this study was to evaluate the antifungal activity of Vitis vinifera byproducts (VIDES) and flesh-coloured potato (FCP) extracts against Rhizoctonia sp. in potato crops. Photosynthetic traits, phenolic profiles, and antioxidant and enzymatic activities were determined. The VIDES extract showed a 151.4% improvement in stomatal conductance and a 258.5% improvement in the photosynthetic rate compared to the plants without infection. Regarding the enzymatic antioxidant activity, the best response was found in the FCP treatments with 30 min of application, with increases of 25%, 161%, and 450% in ascorbate peroxidase, catalase (CAT), and glutathione reductase (GR) activities, respectively, compared to plants without infection. For the VIDES extract, a 15 min application produced an 83% increase in CAT activity, whereas a 181% increase in GR activity compared to plants without infection was produced after a 30 min application. A similar behaviour was observed for antioxidant compounds, where FCP had a higher concentration of compounds and antioxidant activity. This finding suggests that FCP and VIDES promote the synthesis of plant-defence compounds against Rhizoctonia sp. in potato crops, in which the application time is a determining factor.


Introduction
Potato (Solanum tuberosum L.) is one of the most important food crops worldwide [1]. In general, it has a higher source of antioxidant compounds that can reduce the risk of many human diseases [1]. Worldwide, production of this tuber is as high as 380 million tons per year, and a total of 20 million hectares are used in planting, with potato ranking fifth as the main source of energy in the nutrition of the human population [2]. In Chile, approximately fifty thousand hectares are cultivated per year, ranking it as the fourth crop in terms of surface area with the largest number of farmers dedicated to its production, with the region of La Araucanía being the main producer and distributor of potato in the country [3].

Variables and Photosynthetic Performance
Photosynthetic determinations were performed on potato plants. Regarding the performance of photosystem II (ΦPSII), significant differences were observed between the controls, indicating that the ΦPSII efficiency decreased by 4.1% in plants with Rhizoctonia sp. compared to the noninoculated plants. On the other hand, a significant difference was also observed between the treatments with the application of the FCP extract and Vitis vinifera byproducts (VIDES). Treatment with FCP showed better results compared to the treatments with the VIDES extract application ( Figure 1A). For the stomatal conductance (gs: mmol H 2 O m −2 s −1 ), significant differences were observed between the treatments with the application of FCP and VIDES. Specifically, VIDES extract application for 15 min increased the stomatal conductance by 42.7% compared to the FCP application for the same amount of time and by 151.4% compared to plants without Rhizoctonia inoculation ( Figure 1B). The photosynthetic rate (A) also showed significant differences between the controls, where the control with the presence of Rhizoctonia sp. showed a 92.8% lower performance than the treatments with pathogen-free plants. For the treatments with the FCP extract application, no differences were observed between them. In contrast, significant differences were observed among the treatments with the VIDES extract application, with a 28.05% increase in the 15 min application compared to the application of the same extract for 30 min. It is worth noting that RV15 showed significant differences from all treatments, with the highest results for this parameter ( Figure 1C). In terms of the internal concentration of CO 2 (Ci) ( Figure 1D), it should be highlighted that the controls did not show significant differences between them, and the application of the FCP extract for 30 min showed significant differences and the highest value among the treatments, with a 19% increase compared to commercial fungicides observed. Finally, in terms of water use efficiency (WUE) ( Figure 1E), significant differences were observed among the control groups, with the treatment with Rhizoctonia showing a 69.9% decrease compared to the healthy plant treatments. Furthermore, the application of the FCP extract for 15 min achieved similar results as the commercial fungicides, which did not show significant differences from the healthy plant controls and had the highest results for this parameter among the treatments.

Enzymatic Antioxidant Activity in Potato Leaves
Among the treatments, the FCP extract application for 30 min (RF30) showed a higher catalase, glutathione reductase, and ascorbate peroxidase enzymatic activity compared to the treatments with the FCP extract applied for 15 min, VIDES extract applied for 30 and 15 min, and commercial fungicides (Figure 2A-C). Regarding the extracts, the application time of the FCP extract increased the enzymatic activity by 43.1% for catalase (CAT), 160.2% for glutathione reductase (GR), and 28.9% for ascorbate peroxidase (APX) with 30 min of application compared to 15 min of application. On the other hand, in plants treated with the VIDES extracts, the opposite trend was observed for CAT and APX, where the CAT activity decreased by 32.2% and 18.6% in the treatments with a 30 min application time compared to the 15 min application time. In contrast, the VIDES treatment for 30 min produced a 159.3% increase in the GR activity compared to the 15 min application treatment (Figure 2A-C).
With respect to TBARS ( Figure 2D), it is worth noting that RF30 had the lowest value of lipid peroxidation compared to the extract application treatments. Additionally, RF30 had the highest enzymatic activity value. The same trend is observed in the case of the Rhizoctonia-inoculated control (RN), which shows a lower lipid peroxidation compared to the control of healthy plants without inoculation. Consistent with this finding, RN exhibited the highest CAT enzymatic activity value among all treatments, and for GR and APX, it was the second highest compared to all the treatments. . Photosynthetic rate measured in Solanum tuberosum leaves of VR808 genotype inoculated or not with Rhizoctonia sp. and treated with flesh-coloured potato (FCP) extracts, Vitis vinifera (VIDES), or commercial fungicides. The small letters in each value represent the statistically significant difference according to Tukey's test (p < 0.05). In detail, (A) photosystem II quantum yield (ΦPSII) (mmol CO2 μmol −1 photons absorbed); (B) stomatal conductance (gs) (mmol H2O m −2 s −1 ); (C) photosynthesis rate (A) (μmol CO2 m −2 s −1 ); (D) CO2 leaf internal concentration (Ci) (μmol mol −1 ); (E): water use efficiency (WUE) (mmol CO2 mol −1 H2O). Where NR: no Rhizoctonia sp. inoculation, NE: no extract or commercial fungicide application, RF: Rhizoctonia and FCP extract, RV: Rhizoctonia and VIDES extract, 15: application for 15 min, 30: application for 30 min, RM: Rhizoctonia sp. and MONCUT, RX: Rhizoctonia sp. and ReflectXtra, RN: Rhizoctonia sp. and extract or commercial fungicide.

Enzymatic Antioxidant Activity in Potato Leaves
Among the treatments, the FCP extract application for 30 min (RF30) showed a higher catalase, glutathione reductase, and ascorbate peroxidase enzymatic activity compared to the treatments with the FCP extract applied for 15 min, VIDES extract applied for 30 and 15 min, and commercial fungicides (Figure 2A-C). Regarding the extracts, the application time of the FCP extract increased the enzymatic activity by 43.1% for catalase (CAT), 160.2% for glutathione reductase (GR), and 28.9% for ascorbate peroxidase (APX) with 30 min of application compared to 15 min of application. On the other hand, in plants treated with the VIDES extracts, the opposite trend was observed for CAT and APX, where the CAT activity decreased by 32.2% and 18.6% in the treatments with a 30 min application time compared to the 15 min application time. In contrast, the VIDES treatment for 30 min produced a 159.3% increase in the GR activity compared to the 15 min application treatment (  . Photosynthetic rate measured in Solanum tuberosum leaves of VR808 genotype inoculated or not with Rhizoctonia sp. and treated with flesh-coloured potato (FCP) extracts, Vitis vinifera (VIDES), or commercial fungicides. The small letters in each value represent the statistically significant difference according to Tukey's test (p < 0.05). In detail, (A) photosystem II quantum yield (ΦPSII) (mmol CO 2 µmol −1 photons absorbed); (B) stomatal conductance (gs) (mmol H 2 O m −2 s −1 ); (C) photosynthesis rate (A) (µmol CO 2 m −2 s −1 ); (D) CO 2 leaf internal concentration (Ci) (µmol mol −1 ); (E): water use efficiency (WUE) (mmol CO 2 mol −1 H 2 O). Where NR: no Rhizoctonia sp. inoculation, NE: no extract or commercial fungicide application, RF: Rhizoctonia and FCP extract, RV: Rhizoctonia and VIDES extract, 15: application for 15 min, 30: application for 30 min, RM: Rhizoctonia sp. and MONCUT, RX: Rhizoctonia sp. and ReflectXtra, RN: Rhizoctonia sp. and extract or commercial fungicide.

Determination of Phenolic Compounds and Their Relationship with the Antioxidant Activity in Potato Leaves
Four phenolic compounds were detected, corresponding to one HCAD (5-caffeoylquinic acid) and three flavonols (quercetin-3-glucosylrutinoside, quercetin-dihexoside, and quercetinrutinoside) (Figure 3A-D; Table 1). In general, the results show higher concentrations of flavonols and lower concentrations of HCAD. Regarding the individual concentrations of 5-caffeoylquinic acid (pick 1), quercetin-3-glucosylrutinoside (pick 2), and quercetinrutinoside (pick 4), a decrease in concentration was observed, whereas the time of application of the extract increased, which occurred in both extracts, FCP and VIDES. On the other hand, the extract treatments showed the highest values of total phenolic compounds ( Figure 4A) in the treatment with RF15. With respect to TBARS ( Figure 2D), it is worth noting that RF30 had the lowest value of lipid peroxidation compared to the extract application treatments. Additionally, RF30 had the highest enzymatic activity value. The same trend is observed in the case of the Rhizoctonia-inoculated control (RN), which shows a lower lipid peroxidation compared to the control of healthy plants without inoculation. Consistent with this finding, RN exhibited the highest CAT enzymatic activity value among all treatments, and for GR and APX, it was the second highest compared to all the treatments.

Determination of Phenolic Compounds and Their Relationship with the Antioxidant Activity in Potato Leaves
Four phenolic compounds were detected, corresponding to one HCAD (5caffeoylquinic acid) and three flavonols (quercetin-3-glucosylrutinoside, quercetin-dihexoside, and quercetin-rutinoside) (Figure 3A-D; Table 1). In general, the results show higher concentrations of flavonols and lower concentrations of HCAD. Regarding the individual concentrations of 5-caffeoylquinic acid (pick 1), quercetin-3-glucosylrutinoside (pick 2), and quercetin-rutinoside (pick 4), a decrease in concentration was observed, whereas the time of application of the extract increased, which occurred in both extracts, FCP and VIDES. On the other hand, the extract treatments showed the highest values of total phenolic compounds ( Figure 4A) in the treatment with RF15.  In general, the RF15 treatment stands out for showing a significant difference compared to the other treatments in terms of flavonols, HCADs, and total phenols, except for quercetindihexoside, which is only present in the control treatment in the presence of Rhizoctonia ( Figure 3C). In general, the RF15 treatment stands out for showing a significant difference compared to the other treatments in terms of flavonols, HCADs, and total phenols, except for quercetin-dihexoside, which is only present in the control treatment in the presence of Rhizoctonia ( Figure 3C).

Antioxidant Activities
In terms of the application time of the extracts, in the case of the Trolox equivalent antioxidant capacity (TEAC) method ( Figure 4B), the treatments with the FCP and VIDES extracts showed a higher activity with 15 min of application compared to the same extracts applied for 30 min. Specifically, for RF30, the TEAC decreased by 16.08% compared to RF15. For the application of the VIDES extract, the treatment with Rhizoctonia sp. inoculum and application of the grapevine extract for 30 min (RV30) decreased by 20% compared to the application of the grapevine extract for 15 min (RV15). Via the copper-reducing antioxidant capacity (CUPRAC) method ( Figure 4C), a similar trend was observed, where the application of the FCP extract for 15 min (RF15) had the highest value compared to the rest of the treatments. However, there was also a decrease under the application of the FCP extract for 30 min (RF30) compared to RF15. In terms of the antioxidant activity determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method ( Figure 4D), it can be observed that RF30 had a higher activity than RF15, indicating that increasing the application time enhanced the DPPH-determined capacity for FCP. On the other hand, RV30 had a lower activity compared to RV15, indicating the opposite effect, where a shorter application time of the VIDES extract resulted in a higher capacity determined by this method.

Antioxidant Activities
In terms of the application time of the extracts, in the case of the Trolox equiv antioxidant capacity (TEAC) method ( Figure 4B), the treatments with the FCP and VI extracts showed a higher activity with 15 min of application compared to the same ext applied for 30 min. Specifically, for RF30, the TEAC decreased by 16.08% compare RF15. For the application of the VIDES extract, the treatment with Rhizoctonia sp. inocu and application of the grapevine extract for 30 min (RV30) decreased by 20% compar the application of the grapevine extract for 15 min (RV15). Via the copper-reducing oxidant capacity (CUPRAC) method ( Figure 4C), a similar trend was observed, wher application of the FCP extract for 15 min (RF15) had the highest value compared to rest of the treatments. However, there was also a decrease under the application o FCP extract for 30 min (RF30) compared to RF15. In terms of the antioxidant activity termined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method ( Figure 4D), it can b served that RF30 had a higher activity than RF15, indicating that increasing the app tion time enhanced the DPPH-determined capacity for FCP. On the other hand, RV30 a lower activity compared to RV15, indicating the opposite effect, where a shorter a cation time of the VIDES extract resulted in a higher capacity determined by this met

Low-Molecular-Weight Organic-Acid Exudation
Oxalic acid was identified as the only low-molecular-weight acid in the rhizosp of potato plants. It was identified at concentrations ranging from 94.50 ± 10.03 mg kg 331.15 ± 5.9 mg kg −1 ( Figure 5). In detail, RV30 presented the lowest concentration v Rhizoctonia sp. and ReflectXtra, RN: Rhizoctonia sp. and extract or commercial fungicide. Different letters indicate significant differences according to Tukey's test (p < 0.05). Results are given as the mean of the values for each treatment ± the standard deviation.

Low-Molecular-Weight Organic-Acid Exudation
Oxalic acid was identified as the only low-molecular-weight acid in the rhizosphere of potato plants. It was identified at concentrations ranging from 94.50 ± 10.03 mg kg −1 to 331.15 ± 5.9 mg kg −1 ( Figure 5). In detail, RV30 presented the lowest concentration value and a significant difference with RV15 and MONCUT, the latter being the treatments that had the highest concentrations among the treatments. It should be noted that the application time appears to be an important factor in the amount of oxalic acid synthesised and exuded by the plant's roots. Specifically, a decrease of 85.1% was observed in the FCP treatments when the application time increased from 15 to 30 min. The same occurred with the VIDES treatments, where the concentration of oxalic acid decreased by 86.6% when the application time increased from 15 to 30 min.

Agronomic Yield
In terms of the agronomic yield, the mass (g) of the tubers after harvest was determined, with a range of masses between 138 ± 3 g and 185 ± 1 g ( Table 2). The control treatment without Rhizoctonia inoculation (NRNE) had the highest mass compared to all the treatments, and it showed a significant difference compared to the control treatment inoculated with Rhizoctonia (RN). Specifically, RN had an 18.1% lower total tuber mass compared to NRNE. On the other hand, no significant differences were observed between the FCP and VIDES extract treatments. It is worth noting that significant differences in the tuber mass were observed between the treatment with the commercial fungicide ReflectXtra (RX) and the treatments with the extracts. RX had an approximately 20% lower tuber mass than RF15, RF30, RV15, and RV30. and a significant difference with RV15 and MONCUT, the latter being the treatments that had the highest concentrations among the treatments. It should be noted that the application time appears to be an important factor in the amount of oxalic acid synthesised and exuded by the plant's roots. Specifically, a decrease of 85.1% was observed in the FCP treatments when the application time increased from 15 to 30 min. The same occurred with the VIDES treatments, where the concentration of oxalic acid decreased by 86.6% when the application time increased from 15 to 30 min.

Agronomic Yield
In terms of the agronomic yield, the mass (g) of the tubers after harvest was determined, with a range of masses between 138 ± 3 g and 185 ± 1 g ( Table 2). The control treatment without Rhizoctonia inoculation (NRNE) had the highest mass compared to all the treatments, and it showed a significant difference compared to the control treatment inoculated with Rhizoctonia (RN). Specifically, RN had an 18.1% lower total tuber mass compared to NRNE. On the other hand, no significant differences were observed between the FCP and VIDES extract treatments. It is worth noting that significant differences in the tuber mass were observed between the treatment with the commercial fungicide ReflectXtra (RX) and the treatments with the extracts. RX had an approximately 20% lower tuber mass than RF15, RF30, RV15, and RV30.

Principal Component Analysis
A principal component analysis was performed (Figure 6), considering the presence of Rhizoctonia sp., the application of the extracts (FCP and VIDES), the application of the commercial fungicide (MONCUT (M) or ReflectXtra (X)), and the application time (15 and 30 min, and no application (0)). In relation to the components, it was observed that there is an influence of the antioxidant activity on the treatments with the FCP extract, particularly towards RF15. There is also a correlation between GR, CAT, and oxalic acid in RF30; however, all the antioxidant mechanisms reported are correlated with the use of FCP, and the variation between them is determined by the time of application. On the other hand, an opposite tendency occurs in the use of the VIDES extract, and the influence of the Plants 2023, 12, 2974 9 of 17 extract treatments on the photosynthetic parameters is observed. This finding suggests that VIDES may be a growth promoter, not an extract that promotes plant-defence mechanisms. Finally, no direct influence of the components on the controls was observed. However, this component analysis shows a difference in correlation between the control plants, which is significant and demonstrates the influence of Rhizoctonia sp. on the defence mechanisms and development of potato plants. In relation to the fungus, it was observed that the components only had influence on the treatments in the presence of Rhizoctonia sp. In general, it was observed that most of the compounds correlated with the treatments with extract application, suggesting a possible protective action against Rhizoctonia sp. as a promoter of plant-defence mechanisms.
commercial fungicide (MONCUT (M) or ReflectXtra (X)), and the application time (15 30 min, and no application (0)). In relation to the components, it was observed that t is an influence of the antioxidant activity on the treatments with the FCP extract, par larly towards RF15. There is also a correlation between GR, CAT, and oxalic acid in R however, all the antioxidant mechanisms reported are correlated with the use of FCP, the variation between them is determined by the time of application. On the other h an opposite tendency occurs in the use of the VIDES extract, and the influence of the tract treatments on the photosynthetic parameters is observed. This finding suggests VIDES may be a growth promoter, not an extract that promotes plant-defence me nisms. Finally, no direct influence of the components on the controls was observed. H ever, this component analysis shows a difference in correlation between the control pla which is significant and demonstrates the influence of Rhizoctonia sp. on the defence m anisms and development of potato plants. In relation to the fungus, it was observed the components only had influence on the treatments in the presence of Rhizoctonia s general, it was observed that most of the compounds correlated with the treatments w extract application, suggesting a possible protective action against Rhizoctonia sp. as a moter of plant-defence mechanisms.

Discussion
In a study conducted by Cayún et al. (2023) [21] on potato plants of the same genotype used in our study (VR808), it was determined that the ΦPSII values for the control treatment of healthy plants were close to 0.75. On the other hand, plants treated with commercial fungicides (ReflectXtra and MONCUT) showed values close to 0.7, where the decrease between the control and the fungicides was not significant. Regarding the quantum yield of the photosystem II values in our study, in the case of the control without the pathogen presence, identical results to the control study reported by Cayún et al. (2023) [21] were observed. The same applies to the results related to the commercial fungicide treatments, with the difference being that in our case, the decrease in the quantum yield of photosystem II between treatments is significant. On the other hand, the treatment in the presence of Rhizoctonia resulted in a significantly decreased quantum yield of photosystem II compared to the healthy control, reaching an average value of 0.73 mmol CO 2 µmol −1 of absorbed photons. This result is similar to what was reported in a previous study for treatments under MONCUT fungicide, indicating that Rhizoctonia negatively impacts the performance of photosystem II, similar to commercial fungicides.
It is worth noting that there is no existing evidence in the literature regarding the results of photosynthetic parameters under similar conditions to those carried out in the present study. Stomatic conductance (gs) is associated with a higher photosynthetic rate (A) because with stomatal opening, more CO 2 is consumed, decreasing the internal CO 2 concentration in the leaf (Ci). Many factors may mediate this relationship, such as the use of agriculturally beneficial microorganisms like mycorrhizae, resulting in an improved water use efficiency (WUE) and availability for gas exchange, suggesting an improvement in the stomatal conductance and photosynthetic rate [22,23]. Something very similar occurs in our results regarding the use of natural extracts, such as the VIDES extract, which shows results that demonstrate an increase in stomatal opening and a decrease in the internal CO 2 concentration, leading to an increase in the photosynthetic rate of plants under this treatment compared to the controls. It is worth noting that these improvements are not only compared to the control with the Rhizoctonia sp. presence but also compared to the control of healthy plants without pathogens. This finding suggests that the VIDES extract not only functions as a protector against Rhizoctonia but may also act as a promoter of potato plant development. Finally, Wu et al. (2021) [24] reported the negative influence of R. solani on photosynthetic parameters, such as the photosynthetic rate, which is consistent with our results, as the control treatment without application and in the presence of Rhizoctonia sp. presented the lowest values of A among the treatments.
Plant enzymes as part of the antioxidant defence system include superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase, among others. Together, they form a complex mechanism to reduce and eliminate reactive oxygen species (ROS), generating a different reaction mechanism mediated by the type of stress [25]. During a stress situation, SOD catalyses the elimination of O 2 − into O 2 and H 2 O 2 , which CAT and APX convert to O 2 and H 2 O and subsequently eliminate. GR catalyses the reduction of oxidised glutathione to reduced glutathione [26]. The detected trends bring us closer to an explanation of the mechanism of action of the extracts, as in most cases, RF30 surpasses the enzymatic activity of RN. This finding leads us to conclude that antioxidant enzymes act as protectors, preventing the damage to cell membranes caused by Rhizoctonia. In this case, FCP with a 30 min application not only provides protection but also promotes this mechanism, as it shows a higher enzymatic activity and lower lipid peroxidation compared to the Rhizoctonia-inoculated treatment. Youssef et al. (2016) [27] reported that in tomato plants infected with R. solani and using AMF as a control agent, infection by R. solani led to an increase in the response of antioxidant enzymes due to the elevated ROS levels caused by pathogen infection. They observed a significant decrease in the enzymatic activity in the treatments with biocontrol agents, suggesting a positive regulation of antioxidant enzymes in response to Rhizoctonia sp. This finding is consistent with our results regarding enzymatic activity quantification; however, it contradicts the findings from the TBARS analysis. The treatments with a higher enzymatic activity exhibited a lower lipid peroxidation, indicating that the protection system functions preventively or as a defence mechanism. On the other hand, the treatments with a lower enzymatic activity showed a higher lipid peroxidation. Therefore, this positive regulation depends on the mechanism used by the biocontrol agent, which seems to act as an enzymatic promoter for plant defence in the case of the extracts used in our study. It is worth noting that the application time of the extracts appears to be a determining factor in the plant protective action against Rhizoctonia sp. In the case of the FCP extract, as the application time increased from 15 to 30 min, the protective effect was enhanced, as we detected a higher enzymatic activity in RF30 than in RF15 and less lipid peroxidation in the RF30 treatments. Based on these results, it is possible to demonstrate that the extracts have a protective effect on plants under oxidative stress caused by the invasion of a pathogen such as Rhizoctonia sp., as enzyme synthesis increases during exposure to oxidative stress [26,28].
Our results indicate that the systemic response against Rhizoctonia sp. seems to be related to an increase in the synthesis of phenolic compounds in the plant. However, in a study conducted on cucumber plants, an increase in the concentration of 23 phenolic compounds, including HCAD and flavonols, was found in the leaves of plants without systemic symptoms of R. solani disease. This finding suggests that the accumulation of these compounds contributes significantly to protection against the pathogen [29], which is consistent with the results reported in the present study, as the treatments with FCP exhibited higher concentrations compared to the controls, suggesting the important role of the extracts as protective agents and providing insights into their mechanism of action. The results are interesting because phenolic compounds such as quercetin-dihexoside are synthesised in plants that are unprotected, exposed to the pathogen, and likely infected by it. It is worth noting that the common factor in the other treatments is the use of biocontrol agents, such as extracts or commercial fungicides, which validates the efficiency of the in vivo protective action of the extracts against the pathogen. Relatedly, it is suggested that the synthesis of quercetin-dihexoside is a specific defence mechanism and/or indicator against Rhizoctonia, which deserves further study to demonstrate its usefulness and function. Studies suggest that the use of commercial synthetic fungicides has a negative impact on tuber secondary metabolites, decreasing the total concentrations of the reported compounds [10]. While the previous results were determined in tubers, they provide evidence of the negative impact of fungicides, which is consistent with our results in leaves, as the same trend occurs. Likewise, according to Nawrocka et al. (2018) [29], in cucumber plants infected with R. solani and treated with Trichoderma atroviride (TRS25), the systemic defence response in the plant against pathogen infection was strongly related to the improvement in the synthesis of phenolic compounds, increasing the synthesis of flavonols and hydroxybenzoic acids.
The highest antioxidant activity was detected in the CUPRAC assay. It has been reported that the antioxidant activity responded better to phenolic compounds such as flavonols and HCADs [30]. This finding agrees with other studies where the CUPRAC antioxidant activity increased in plants inoculated with AMF, given its high concentration of flavonols and HCADs [11]. On the other hand, Ruiz et al. (2019) [31] reported that strawberry fruits presented a higher antioxidant activity as observed using the TEAC method, which is explained by the TEAC method showing better antioxidant activity results in the presence of anthocyanins compared to flavonols and HCADs. Finally, in potato leaves of the genotype VR808, Fritz et al. (2022) [11] reported an improvement in the antioxidant activity in plants inoculated with AMF. These symbiotic microorganisms present a protective and promoting role of the antioxidant activity, as do the extracts in the present study. On the other hand, Kang et al. (2015) [32] showed that R. solani inhibited Chinese cabbage plant growth, had effects on the photosynthetic parameters, and, in addition, increased the content of the total phenols and antioxidant capacity as determined by the DPPH method. In tomato plants, R. solani infection reduced the chlorophyll and anthocyanin contents in the leaves but increased the total phenol and flavanol contents [33]. It is important to note that Fritz et al. (2022) [11] noted a significant decrease in the antioxidant activity in potato plants using ReflectXtra fungicide without the presence of pathogens. This result suggests that the use of fungicides decreases the plant's self-defence response, and the above reaffirms the idea that these extracts are beneficial for potato plants in the presence of Rhizoctonia sp., as they promote the synthesis of antioxidant compounds, increasing their accumulation in the plant and, in turn, the antioxidant capacity [29].
Oxalic acid induces plant resistance to R. solani by enhancing defence enzymes and related compounds [34]. Therefore, the exudation of organic acids is not only a solubilisation mechanism but also a defence mechanism against pathogens. Parada et al. (2019) [35] reported higher concentrations of oxalic acid exudates in strawberry plants inoculated with AMF than in our study, suggesting that infection by Rhizoctonia sp. influences oxalic acid exudation by decreasing this defence mechanism; however, extracts such as FCP can enhance that mechanism by promoting its exudation.
Regarding the agronomic yield, in the study conducted by Alarcón et al. (2022) [10], the reported biomass of tubers in the VR808 genotype differs from our results in this study. In their case, the control group showed a total biomass of approximately 280 g, while our results show a decrease of 100 g in the biomass of tubers in the control group of healthy plants. This variation may be attributed to the seasonal differences and climatic conditions that the crops faced. It is important to note that the same fertilisation was used in both studies. On the other hand, we identified similarities between our results and those of the treatment with the fungicide MONCUT, indicating that this fungicide had a similar effect in both studies. However, a significant difference was observed in the treatment with the fungicide ReflectXtra, where we reported a lower biomass of tubers compared to the study by Alarcón et al. (2021) [10]. This finding suggests that the presence of the pathogen had a negative impact on our crops, even when commercial fungicides were used. These results support all the previously reported metabolomic results, as it is observed that the pathogen and commercial fungicides have a negative impact on tuber production, whereas the applied extracts protect the plant and yield favourable results similar to those of healthy plants.
Finally, the active compounds of the extracts are related to the physiological and biochemical responses of the plants, and some of the positive effects on plants include increased photosynthetic activity, consumption and efficient use of water, and antioxidant capacity, among others [36]. However, the application time had a similar trend to the difference in extracts; the shorter application time correlated with the antioxidant capacity, and as the time increased, it correlated with the photosynthetic parameters.

Extract Preparation
Flesh-coloured potatoes (CB2011-104 genotype) were provided by Novaseed Ltd.a. (Puerto Varas, Chile). The extraction procedure was carried out according to the description by Ercoli et al. (2021) [18], with modifications. In detail, 500 g of mashed potato was mixed with 1000 mL of extraction (15% glacial acetic acid in absolute ethanol, v/v). Subsequently, ultrasound was applied for 60 s at 40% amplitude, and the mixture was left in an orbital shaker at 200 rpm for 10 min for homogenisation. Then, it was centrifuged for 10 min at 4 • C and 3000× g. The procedure was performed only once and stored at −20 • C in darkness for further treatment. The VIDES extract was made from grape canes of Vitis vinifera cv. Pinot noir and was provided by the University of Concepción (Concepción, Chile). Its preparation was carried out according to the description by Escobar et al. (2021) [37], in a 750 L stainless-steel reactor using an ethanol/water solution (80:20 v/v). Afterwards, it was stored in darkness at 4 • C.
After the preparation of the extracts, it was necessary to select a maintenance solvent without antifungal activity against Rhizoctonia sp. based on their properties of solubilising and maintaining the stability of the phenolic compounds in the extracts. In detail, the FCP extract was rotary evaporated and resuspended in a 0.05% v/v Tween 20 solution. For the VIDES extract, the same procedure was performed, but it was resuspended in dimethyl sulfoxide (DMSO).

Biological Material and Experimental Design
This study was conducted in an outdoor greenhouse with the temperature and light/dark photocycle according to seasonal environmental conditions between December 2021 and February 2022 in the Departamento de Ciencias Químicas y Recursos Naturales of the Universidad de La Frontera, Temuco, Chile, with average temperature highs around 25 • C and average lows around 10 • C. Additionally, the region encountered increased precipitation, with rainy days alternating with sunny periods, and relative humidity ranging between 60% and 80%. A completely randomised design with five replicates per treatment was carried out. The main factors consisted of (i) inoculation of Rhizoctonia sp. and a control without inoculation; (ii) application of extracts (VIDES and FCP), commercial fungicides (ReflectXtra (X) or MONCUT (M)), and a control without application; and (iii) time of application of extracts (15 or 30 min) in addition to a control without application ( Table 3). The potato genotype used was VR808 (white-fleshed), also provided by Novaseed Ltd.a. In terms of the factors, the following treatments were created: control without inoculation of Rhizoctonia sp. For this obtention, a specific isolation for Rhizoctonia sp. was performed from a tuber displaying symptoms caused by this pathogen's disease. On the other hand, the strains were stored through long-term preservation, cryopreservation with glycerol and mineral oil at −80 • C. Before establishing the greenhouse trial, the available Rhizoctonia sp. mycelium, obtained from PDA culture with 3-day growth at 28 • C, was inoculated onto 50 potato seeds that were used for planting. Previously, fungal density was determined by the method described by Borie & Rubio (2003) [38]. After inoculation, 10 seeds were immersed in FCP extract, 5 of them for 15 min and the remaining ones for 30 min. In turn, 10 seeds were immersed in grapevine extract, 5 of them for 15 min and the remaining seeds for 30 min. On the other hand, 5 seeds were treated with the commercial fungicide MONCUT, and 5 seeds were treated with the commercial fungicide ReflectXtra, in both cases according to the supplier's instructions.
For sowing, 11 L pots and peat substrate were used (raw material brown peat Sphagnum H2-H4 von Post * 10% Perlite, pH 5.5). In addition, KH 2 PO 4 and urea were added as fertiliser [3]. The plants were watered as needed. Day 120 marked the collection of data and photosynthetic parameters using the second-youngest leaf. Harvesting of fresh biomass took place the following day, preserving the same leaves used for data collection by immersion in liquid nitrogen, followed by transfer to a temperature of −80 • C for subsequent analysis. Additionally, the resulting tubers from each treatment were harvested, and their fresh biomass and tuber numbers by pot were determined.

Enzymatic Antioxidant Activity and Thiobarbituric Acid-Reactive Substances
For enzymatic extracts, 0.3 g samples of leaf tissue were frozen in liquid nitrogen and then ground in 0.9 mL of solution containing 0.1 mol L -1 phosphate buffer (pH 7.0) and 2.5% (w/v) polyvinylpyrrolidone [21]. The supernatant obtained was used for protein quantification via the Bradford method [39]. The enzymatic determination of catalase, ascorbate peroxidase, and glutathione reductase was performed according to the description by Aguilera et al. (2020) [40]. A microplate spectrophotometer was used for all determinations (Synergy HTX UV-visible Biotek, Winooski, VT, USA).
Lipid peroxidation was determined by malondialdehyde (MDA) formation using the thiobarbituric acid method according to the description by Du & Bramlage (1992) [41] with modifications. After extraction, 300 µL of supernatant was taken, and 1.2 mL of a solution containing 20% TCA and 0.5% TBA was added. The mixture was incubated at 95 • C on a hot plate for 30 min, after which time it was removed and cooled in an ice bath and then centrifuged at 10,000× g for 10 min at 4 • C. Finally, absorbance readings were taken at 440, 532, and 600 nm in a Synergy HTX UV-visible microplate spectrophotometer Biotek (Winooski, VT, USA).

Identification and Quantification of Phenolic Compounds in Leaves
The extraction and identification of phenolic compounds was carried out according to the description by Fritz et al. (2022) [11]. High-performance liquid chromatography with diode array detection (HPLC-DAD) equipment Shimadzu (Tokyo, Japan) equipped with a quaternary pump (LC-20AT), a degassing unit (DGU-20A5R), a column oven (CTO-20A), an autosampler (SIL-20A), and a UV-Vis diode array detector (SPD-M20A) was used for identification and quantification using a Kromasil C 18 column (100 × 4.6 mm, 2.5 µm) with a Nova-Pak C 18 precolumn (22 × 3.9 mm, 4 µm; Waters) at 40 • C. HCADs were quantified at 320 nm and flavonols at 360 nm using chlorogenic acid and quercetin as external calibration standards, respectively. Identity assignments were performed according to the description by Nova et al. [42] using an HPLC-DAD-QTOF-MS/MS Compact (Bruker Daltonics GmbH, Bremen, Germany). Instrument control and data collection were carried out using Compass DataAnalysis 4.4 SR1 (Bruker Daltonics GmbH).

Determination of Total Phenolics and Antioxidant Activity
The determination of total phenols, Trolox equivalent antioxidant capacity (TEAC), copper-reducing antioxidant capacity (CUPRAC), and antioxidant activity by the 2,2diphenyl-1-picrylhydrazyl (DPPH) method was performed as described by Parada et al. (2019) [35] in the extracts used for phenolic compound determination, using chlorogenic acid as a standard for the quantification of total phenols. On the other hand, the Trolox standard was used for the TEAC, CUPRAC, and DPPH methods. Measurements were performed on a Synergy HTX UV-visible microplate spectrophotometer (BioTek, Winooski, VT, USA) at 750 nm for the determination of total phenols, 734 nm for TEAC, 450 nm for CUPRAC, and 517 nm for DPPH.

Exudation of Low-Molecular-Weight Organic Acids
Low-molecular-weight organic-acid (LMWOA) concentrations were determined according to the method described by Parada et al. (2019) [35] with modifications. Rhizosphere samples (0.5 g) were dissolved in 10 mL of 0.2 M calcium chloride, then centrifuged at 4000× g for 15 min at 4 • C, and the supernatant was retained. Chromatographic analysis was carried out using HPLC-DAD with characteristics described by Parada et al. (2019) [35]. In detail, a Kromasil C 8 column (250 × 4.6 mm, 5 µm) and a Kromasil C 8 precolumn (100 × 4.6 mm, 5 µm) were used with 0.2 N phosphoric acid, pH 2.1, as the mobile phase at a flow rate of 1.0 mL min −1 in an isocratic system at 30 • C. Quantification was performed at 210 nm by external calibration with oxalic acid as a standard.

Statistical Analysis
All statistical analyses were performed and figures were prepared in R version 4.2.1. One-way ANOVA was used to test for significant differences between measurements of each experimental variable. For the variables with significant differences, the means were compared using the Tukey HSD multiple range test with the package "agricolae" v. 1.3.5. Moreover, the dataset was subjected to principal component analysis (PCA). Confidence ellipses (group means) by differences in treatments, fungus, extract, and time were also generated using the packages "FactoMineR" v. 2.7 and "factoextra" v. 1.0.7.

Conclusions
In detail, the FCP extract increases the concentration of compounds related to the antioxidant defence in the leaves of potato plants for the control of Rhizoctonia sp. On the other hand, the VIDES extract primarily enhances the photosynthetic parameters of potato plants in those artificially infected with Rhizoctonia sp. The above suggests that the extracts exhibit differentiated activities. FCP appears to be associated with the synthesis of plant-defence compounds, whereas VIDES enhances the processes of the plant's primary metabolism, potentially leading to an increase in the plant's growth rate as it improves photosynthetic processes.
The results highlight the positive protective effects of the extracts against the pathogen, which are validated through their agronomic performance. However, it is important to investigate the mechanism employed by the extracts for crop protection, as well as to optimise the type and timing of application. Such work will lead us to the development of applied science and an efficient, useful, and marketable product. It seems that the action of the extracts is mediated by these variables, as well as by the plant-extract relationship that can be established depending on the applications.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.